Herein is reported a cellular FRET assay for the determination of the simultaneous binding of a bispecific antibody to both of its targets/antigens.
The determination of functional binding of antibodies is a routine test made in drug development.
Fluorescence Resonance Energy Transfer (FRET) is a physical phenomenon first described over 50 years ago. FRET is based on the radiationless transfer of energy from a donor molecule to an acceptor molecule. No direct contact of both molecules is required therefore, i.e. it is a distance-dependent energy transfer. Thus, FRET can be used to investigate molecular interactions.
In US 2015/024410 is directed to an in vitro method for determining the binding of an antibody to an Fc receptor expressed in cell membranes or intact cells present in a measurement medium, comprising the following steps: (i) direct or indirect labeling of said Fc receptor with the first member of a pair of FRET partners, or else introducing, into the medium, cell membranes or intact cells whose Fc receptors were labeled beforehand directly with the first member of a pair of FRET partners; (ii) adding said antibody, labeled directly or indirectly with the second member of said pair of FRET partners, to the measurement medium; (iii) measuring the FRET signal, the existence of a FRET signal being representative of the binding of the antibody to the Fc receptor.
A method of measuring the binding activities of an antibody to both an antigen or antigen epitope and an Fc receptor or a fragment thereof, comprising: a step of mixing the antibody with the antigen or antigen epitope labeled with one member of a set of donor and acceptor capable of fluorescent resonance energy transfer and the Fc receptor or fragment thereof labeled with the other member of said set of donor and acceptor; a step of irradiating the resultant mixture with light having a wavelength capable of exciting the donor; and a step of measuring the fluorescence level of said mixture is reported in US 2010/190266.
In US 2006/165685 bispecific anti-Erb-B antibodies and their use in tumor therapy are reported.
In US 2015/0204847 a high-throughput, high-precision method for detecting protein structural changes in living cells is reported. Large stokes shift dyes are reported in US 2015/0090936. In US 2015/0044690 FRET measurement device and FRET measurement method are reported.